A mesenchymal stem cell is one type of somatic stem cells and present in tissues of bone marrow or the like. A mesenchymal stem cell is known as a stem cell having a pluriopotency to differentiate into an adipose cell, a bone cell, a chondrocyte, or the like, and having a self-propagating potential. Currently, the mesenchymal stem cell is used as a cell for transplantation in regenerative medicine field. The mesenchymal stem cell is applied to various diseases such as bone defect, cartilage defect, periodontal disease, myocardial infarction, refractory cutaneous disease, osteoporosis, osteoarthrosis, spinal cord injury, hematopoietic support, and antirejection in organ transplantation. It is expected that the mesenchymal stem cell will be applied to more and more diseases in the future (for example, cerebral infarction, arteriosclerosis obliterans, kidney disorder, and the like).
The mesenchymal stem cell is present in tissues of bone marrow, periostea, or the like. Mesenchymal stem cells taken from such tissues are proliferated and further differentiated into intended cells, so that tissues that can be used in tissue regeneration medicine are prepared. However, since the number of mesenchymal stem cells present in living tissues is little, the use of the mesenchymal stem cells for transplantation requires sufficient proliferation of the cells taken from tissues.
Generally, culturing of animal cells is carried out with the use of a culture medium to which 5 to 20% nonhuman animal-derived serum such as fetal bovine serum or the like is added. The serum is used as a nutrient source for promoting in vitro cell growth and/or proliferation, or a resource for a biologically active substance such as hormone or the like. However, serum is very expensive, and components of serum differ lot by lot because the serum is a natural product. Moreover, it is necessary to purify cultured cells by removing serum-derived proteins or the like from the cultured cells, thereby causing a complicated process. Furthermore, there is a risk that cultured cells are infected with unknown pathogen (such as virus, pathological prion, or the like) that is contained in serum.
Meanwhile, techniques for culturing animal cells without using nonhuman animal-derived serum have been developed. For example, culture of cells used for autologous transplantation treatment (in which cells taken from a patient are cultured and the cultured cells are transplanted to the patient) uses autologous human serum obtained from the same patient. This avoids contamination of cultured cells. However, large amounts of blood are required for producing serum, which gives heavy burden to patients.
In order to avoid the problems, a culture medium that contains no serum (serum-free culture medium) or a culture medium whose content of serum is low (low-serum culture medium) has been developed. Low serum concentration in a culture medium decreases a proliferation ability of cells remarkably decreases or kills the cells. From this reason, in order to produce a culture medium which is capable of culturing cells without losing its proliferation property, it is necessary to add in a culture medium a cell growth factor alternative to serum. Conventionally, a variety of peptide hormones, growth factors or the like are used as a cell growth factor alternative to serum (for example, see Patent Documents and 2). As such a serum-free culture medium, for example, a serum-free culture medium, which uses a HAM's F12 culture medium as a basal medium, and in which insulin, transferrin, and the like is added, is known.
Another known method is a method in which chondrocyte to be used in medical treatment is cultured in a serum-free culture medium in which a fatty acid is added in addition to a growth factor (for example, see Patent Documents 3 and 4). Furthermore, Patent Document 5 discloses a method for culturing a neural stem cell for long periods and its composition.
[Patent Document 1]
Japanese Unexamined Patent Publication, Tokukaihei, No. 8-308561 (published on Nov. 26, 1996)
[Patent Document 2]
Japanese Unexamined Patent Publication, Tokukaihei, No. 9-191874 (published on Jul. 29, 1997)
[Patent Document 3]
Japanese Translation of PCT international application, Tokuhyo, No. 2005-515777 (published on Jun. 2, 2005)
[Patent Document 4]
Japanese Translation of PCT international application, Tokuhyo, No. 2002-529071 (published on Sep. 10, 2002)
[Patent Document 5]
Japanese Translation of PCT international application, Tokuhyo, No. 2003-516141 (published on May 13, 2003)